The presence of a restriction-modification (RM) system represents a barrier for the transformation of bacteria with recombinant DNA. This is especially applicable to Clostridia sp. The recalcitrance of Clostridia to accept foreign DNA is caused by the presence of a potent restriction system, especially the type II restriction endonucleases.
The first breakthrough in the genetic engineering of Clostridia was the modification of transforming DNA with a phage methyltransferase to protect transforming DNA from digestion before electroporation into C. acetobutylicum ATCC 824 (see, e.g., Lee et al., Ann. NY Acad. Sci., 665: 39-51 (1992)). Since then, methylation of DNA prior to transforming Clostridia has become a standard procedure (see, e.g., Mermelstein et al., App. Environ. Microbiol., 59: 1077-1081 (1993); and Tardif et al., J. Ind. Microbiol. Biotechnol., 27: 271-274 (2001))). Although plasmid DNA could be introduced into Clostridia, the transformation efficiency was very low. As a consequence, gene knockout via homologous recombination mechanisms using non-replicative plasmids or linear DNA was very tedious to achieve. The difficulty of targeted gene disruption in solventogenic Clostridia is well illustrated by the low number of mutants published to date.
More recent techniques that rely on anti-sense RNA technology (Scotcher et al., J. Bacteriol., 187: 1930-1936 (2005)) or group II intron (Heap et al., J. Microbiol. Methods, 70: 452-464 (2007)) are very complex and still necessitate methylating the transforming DNA a priori.
There remains a desire for an improved method for stably integrating a nucleic acid sequence of interest into bacteria, especially Clostridia. 